• This is a gliding motility assay using kinesin-1 and rhodamine labeled microtubules.

• The buffer used was PEM (also known as BRB80) with the inclusion of 22063mM D2O.

• Images were taken with an Andor Luca EMCCD camera on an Olympus IX-71 microscope using a 60x 1.42NA PlanApo objective.

• This fileset is replicate 2/3 and ROI 2/15.

• Images were taken to investigate speed variations of microtubules due to the inclusion of D2O in the motility assay.

• The temperature of the slide was held constant at 32°C by an objective heater. Early ROIs, i.e. 1-5, have slower giding speeds due to the increasing temperature of the slide which, was prepared at room temperature before observation. After ROI 5 the gliding speed stabilizes.

• Pixels lengths were measured to be 166nm/pixel using a stage micrometer.

• After the files have downloaded, a simple command line entry will convert the images to a movie. Mencoder must be installed. ls > list.txt | mencoder mf://@list.txt -mf w=496:h=658:fps=30:type=png -ovc lavc -lavcopts vcodec=mpeg4:mbd=2:trell -o output.avi